Project Summary The goal of this project is to develop a novel immunologically designed probiotic therapy to inhibit gut inflammatory processes for the treatment of inflammatory bowel disease (IBD). Over 3 million adults in the U.S. suffer from IBD, an umbrella term encompassing two chronic inflammatory diseases of the gastrointestinal tract: Crohn?s disease (CD) and ulcerative colitis (UC)6. IBD is typically diagnosed in the second or third decades of life; it is life-long, and there is no cure. Current IBD treatments are systemic and can have serious side-effects. Novel therapies that are safe and effective, particularly restoring the natural interaction between the immune system and gut microbiome, are needed and would be life-changing for patients. Intestinal immune regulatory signals tightly govern healthy gut homeostasis, and their breakdown may result in IBD38. The human microbiome, harboring trillions of bacteria, is a critical regulator of these mechanisms. Commensal bacteria function to maintain intestinal epithelial barrier integrity and regulate innate and adaptive immune cell function39. Lactobacillus (L.) acidophilus, a common bacterial species sold as a probiotic that ?promotes immune health?, contains unique surface layer proteins (Slps), including SlpA, SlpB, SlpX, and lipoteichoic acid (LTA)40-43. These Slps interact with pattern recognition receptors (PRR; e.g., C-type lectin receptors) expressed on innate intestinal cells to fine-tune immunity in steady state and disease conditions42. Recently, our research team demonstrated that SlpA binding to the C-type lectin Specific Intracellular adhesion molecule-3 Grabbing Non-integrin homolog-Related 3 (SIGNR3) receptor expressed on dendritic cells lining the gut prevents experimentally induced colitis in multiple models3. Oral delivery of SlpA reduced inflammatory cytokines, strengthened the mucosal membrane barrier, and supported a healthier microbiota make-up in animal models of gut inflammation. In contrast, the effects and protection were not observed in Signr3-/- mice, suggesting that SlpA interaction with SIGNR3 plays a key protective role in regulating the disease condition3. Our goal is to develop R-3750, a SlpA-expressing, thymidine-dependent L. lactis strain, as a novel, orally administered probiotic that functions in IBD to reduce gut inflammation, improve gastrointestinal mucosal barrier function, and restore the natural microbiome make-up. L. lactis provides two key advantages as a delivery vehicle for conveying SlpA to the gut; namely, it has already been safely used in human clinical trials in a genetically manipulated form1, 4 and it does not express any native Slps but can be engineered to selectively overexpress SlpA. This Phase II SBIR application is intended to build upon success of the Phase I and advance R-3750 towards clinical testing. The specific Aims are: 1) optimize R-3750 upstream process development to support manufacturing, 2) create GMP master cell bank, 3) manufacture R-3750 for animal dosing/biomarker and GLP toxicology studies, and 4) complete R-3750 dose optimization and biomarker- based PD studies in mice.